ABSTRACT: African-American (AA) women are more likely to have aggressive tumors and poorer survival than European-American (EA) women. Understanding the biological mechanisms underlying this disparity is a critical unmet need with major public health implications. The tumor immune milieu may play a pivotal role in breast cancer disparities, specifically tumor infiltrating lymphocytes (TILs) that have roles in oncogenesis and disease progression. Using H&E stained slides from a large epidemiological case-control study, we have discovered that AA women have significantly higher levels of TILs than EA women ? especially in the estrogen receptor (ER) negative subgroups. This indicates a distinct biological difference in the tumor immune response related to race and ER status, and also presents a conundrum in which TILs that are often associated with better outcome are higher in AA women, who typically fare worse with breast cancer. These findings indicate that the specific composition of immune cell populations and their spatial arrangement within the tumor environment are likely to be important mitigating factors for the role of TILs in breast cancer progression, and tumor differences between AA and EA women. Our preliminary data from the NanoString PanCancer Immune Panel support this idea by revealing that AA women have gene expression signatures indicative of higher levels of exhausted T cells than EA women, which have a diminished capacity to destroy tumor cells, consistent with the poorer survival observed in AA women. Here, immune gene expression profiles also varied by ER status, pointing to a potential immunoregulatory role of ER in breast cancer. To date, most studies that examine TILs do so in cohorts of predominantly EA women and in-depth immune profiling studies in large cohorts of AA women are lacking. To address this shortcoming, we will employ multispectral staining and imaging with the Vectra Quantitative Pathology System to delineate immune cell populations and ER expression in 996 breast cancer samples from the Women's Circle of Health Study. Building on our preliminary data, we propose to evaluate two immunofluorescence panels, each with multiple biomarkers in their spatio-morphological context. While both panels will include ER and a differentiating cytokeratin marker, panel 1 will include four common T cell markers (CD3, CD4, CD8, FOXP3), and panel 2 will include markers of T cell exhaustion (TIGIT, LAG3, PD-1) that may play a key role in the differential immune response between AA and EA women. Our proposal involves the full implementation of a new generation of biomarker assessment techniques ? multiplexed staining, multispectral imaging, automated scoring, and digital spatial analyses. A thorough comparison of the immune landscape in breast tumors in regards to race and ER expression will inform our understanding of immune escape mechanisms, and how these processes differ by ancestry. This will be critical for designing immunotherapy strategies that will equally benefit AA women, serving to reduce racial disparities in breast cancer.